Journal: Frontiers in Oncology
Article Title: Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-β1-NF-κB-MMP9 axis
doi: 10.3389/fonc.2024.1371307
Figure Lengend Snippet: Identifying TRAF4 and TAK1 on the integrin-β1 signal cascade that links to NF-κB activation. (A) Schematic representation of the NF-κB activation flow via canonical and noncanonical pathways. (B, C) As indicated, the prepared cell lysates of HEK293T transfectants with different combinations of the plasmids were divided into three aliquots. The first was used as the whole cell lysate, i.e., as input (rightmost). The second was used to conduct immunoprecipitations with anti-HA tag antibody-conjugated agarose beads (middle). The remaining third was used to perform similar immunoprecipitations with anti-DYKDDDDK tag antibody-conjugated agarose beads as a negative control for the immunoprecipitation experiment (leftmost). After immunoprecipitation, the expressed products with beads-bound foreign proteins were analyzed by WB with an anti-HA, anti-Myc, or anti-albumin antibody. (D) The indicated cell extracts were incubated with the biotinylated (bio)-integrin-β1 antibody to capture the intrinsic integrin-β1 of the individual cells. On the other hand, bio-control IgG was used as a negative control for the same immunoprecipitation experiment. The captured integrin-β1 was collected by the pull-down method with avidin beads, and the precipitates were further subjected to WB with the indicated antibodies.
Article Snippet: The antibodies used were as follows: mouse anti-HA tag antibody (clone 6E2; Cell Signaling Technology, Danvers, MA), mouse anti-Myc tag antibody (clone 9B11; Cell Signaling Technology), rabbit anti-human MMP9 antibody (Cell Signaling Technology), rabbit anti-human Akt antibody (Cell Signaling Technology), rabbit anti-human phospho (T308)-Akt antibody (Cell Signaling Technology), rabbit anti-human phospho (S473)-Akt antibody (Cell Signaling Technology), rabbit anti-human NF-κB antibody (Cell Signaling Technology), mouse anti-human phospho (S536)-NF-κB antibody (Cell Signaling Technology), rabbit anti-human IKKα antibody (Cell Signaling Technology), rabbit anti-human IKKβ antibody (Cell Signaling Technology), rabbit anti-human phospho (S176/180)-IKKα/β antibody (Cell Signaling Technology), mouse anti-human Iκβα antibody (Cell Signaling Technology), rabbit anti-human phospho (S32)-Iκβα antibody (Cell Signaling Technology), rabbit anti-human integrin-β1 antibody (Proteintech), rabbit anti-human TRAF4 antibody (Proteintech), rabbit anti-human TAK1 antibody (Cell Signaling Technology), rabbit anti-DsRed (RFP) (Takara Bio USA, Mountain View, CA), rabbit anti-GFP (Thermo Fisher Scientific), mouse anti-β-actin (Merk Sigma-Aldrich), and rabbit anti-human albumin (Agilent’s DAKO, Glostrup, Denmark) that cross-reacts with bovine albumin with high affinity in WB.
Techniques: Activation Assay, Negative Control, Immunoprecipitation, Incubation, Avidin-Biotin Assay
Proteintech is a verified supplier 
